PROJECT SUMMARY The goal of this proposal is to develop and evaluate a novel Magnetic Resonance Imaging (MRI) technique, Magnetization Rotation Transfer (MRT), that can provide information on two types of molecular changes within tissues simultaneously, and which may be used for quantitative tissue characterization. MRT can map variations within tissue of both mobile proteins and specific immobile metabolites. Images that reflect MRT can be used for the diagnosis and assessment of various pathologies and will have immediate translational application for the assessment of solid tumors. Conventional Magnetization Transfer (MT) between solute molecules and water has previously been extensively exploited to report the effects of off-resonance preparation radio-frequency pulses on water, but discriminating specific molecular MT effects in biological tissue has been challenging because of the presence of lipids, asymmetric background lineshapes and overlapping resonances. This proposal aims to address these deficiencies by developing a variation of MT imaging, MRT, which is a more robust method compared to conventional MT imaging because it is feasible to isolate more specific MT effects and it is less prone to artifacts and the influence of experimental variables. MRT involves the subtraction of two signals acquired with pulsed-MT sequences at different irradiation flip angles but the same average power. By this strategy, contributions from spins with extremely short T2 (e.g. macromolecules), relatively fast exchange rates with water (e.g. amine protons), and direct water saturation effects of off-resonance irradiation will be removed. MRT images are therefore sensitive only to spins with relatively slow exchange rates with water (e.g. amide protons or Nuclear Overhauser Enhancements). In our previous studies, we have found two particular contrasts (the MRT effect at 3.5 and -1.6 ppm relative to water resonance) are of special interest. We hypothesize that: (1) MRT(3.5) reflects the mobile protein content of tissues and is more specific than the conventional APT; (2) MT(-1.6) reflects immobile metabolites corresponding mainly to restricted choline-containing compounds. Non-invasive measurements of the mobile protein content and choline-containing metabolites provide a unique approach for tissue characterization, as well as monitoring the status of cancer. In Aim 1, we will further develop and optimize practical MRT imaging methods. Aims 2 and 3 will establish the specificity of MRT(3.5) to mobile proteins in tumors, and investigate the origin of MRT(-1.6) from immobile metabolites, by biochemical and proteomic assays. Overall, the proposed research will extend the capabilities of a ubiquitous imaging technology, MRI, to provide a means for quantitatively characterizing specific molecular contents of tissues in new ways that can immediately be translated into clinical practice.